HIV envelope (Env) protein deposits the nucleocapsid into a host cell by mediating fusion between the viral and cell membranes. Fusion is promoted through conformational changes in Env, which are triggered upon sequential interactions with CD4 and coreceptors. The intermediate steps of HIV fusion that can be targeted by drugs and antibodies are not fully understood, owing to the limited structural information on Env and to the lack of real-time techniques to monitor HIV-cell fusion. An additional complication is that the infectious entry pathways of this virus are not well defined. Whereas HIV is thought to infect cells by fusing directly with a plasma membrane, an accumulating body of evidence suggests that infection can also occur via pH- independent fusion with endosomal compartments. This project focuses on examining HIV entry pathways and on delineating the progression of fusion through intermediate stages. Virus-cell fusion will be directly monitored by the viral core-associated enzyme delivery assay. Individual steps of HIV-cell fusion will be dissected by time-of-addition experiments, using a panel of entry inhibitors targeting CD4 and coreceptor binding steps, as well as the membrane fusion step itself. The contribution from intracellular fusion will be examined by blocking virus endocytosis or by inhibiting both exoplasmic and endoplasmic fusion pathways. Time-resolved imaging of single virus-cell fusion will also be employed to elucidate pathways of HIV entry. Our imaging data suggest that fusion pores formed by individual HIV particles at the cell surface do not fully enlarge, whereas those formed with endosomal membranes dilate efficiently, permitting the release of viral content into the cytosol. These differences in the propensity of fusion pores to dilate have led to the hypothesis that HIV has adapted to form relatively small pores at designated sites, but relies on endocytic machinery to enlarge these pores and initiate infection. In order to test this hypothesis, the formation and enlargement of Env-induced fusion pores at the cell surface and in endosomes will be examined and the roles of viral and cellular factors in dilating these pores will be evaluated. The proposed approaches will help delineate the pathways of HIV fusion that culminate in the release of viral nucleocapsid and will elucidate key intermediate steps of this process. PUBLIC HEALTH RELEVANCE: Human immunodeficiency virus (HIV) initiates infection by depositing its genome into a host cell - a process that involves fusion of the membrane surrounding the viral core with a cell membrane. Membrane fusion is a complex multi-step reaction mediated by specialized HIV envelope protein (Env). To elucidate the mechanism of Env-induced membrane fusion, single HIV particles will be visualized by time-resolved fluorescence microscopy and their fusion with a host cell will be monitored from its initiation (a local merger of viral and cellular membranes) to completion (release of the viral genome into the cytosol). These studies will help define the entry mechanisms of HIV and will suggest new strategies to prevent infection.